Cancer 10: Apoptosis

Question 1

Why does apoptosis occur?

Answer 1

1. Harmful cells (e.g. cells with viral infection, DNA damage).
2. Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens).
3. Excess / unnecessary cells:
   (embryonic development: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs).
4. Obsolete cells (e.g. mammary epithelium at the end of lactation).
5. Exploitation - Chemotherapeutic killing of cells.

Question 2

Define necrosis?

Answer 2

unregulated cell death associated with trauma, cellular disruption and an inflammatory response

Question 3

Define apoptosis?

Answer 3

(programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response

Question 4

What are the features of a cell in necrosis?

Answer 4

Plasma membrane becomes permeable

Cell swelling and rupture of cellular membranes

Release of proteases leading to autodigestion and dissolution of the cell

Localised inflammation

Question 5

What are the two phases in apoptosis?

Answer 5

Latent phase – death pathways are activated, but cells appear morphologically the same

Execution phase –

• Loss of microvilli and intercellular junctions
• Cell shrinkage
• Loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer leaflet)
• Chromatin and nuclear condensation
• DNA fragmentation
• Formation of membrane blebs
• Fragmentation into membrane-enclosed apoptotic bodies

Plasma membrane remains intact - no inflammation

Question 6

What happens to the DNA in apoptosis?

Answer 6

Forms DNA ladders - fragmentation (agarose gel)

Question 7

Define apoptosis-like PCD

Answer 7

some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis

Question 8

Define necorosis-like PCD

Answer 8

Variable features of apoptosis before cell lysis; “Aborted apoptosis”

Question 9

What is the mechanism of apoptotic cell death?

Answer 9

1) The executioners – Caspases

2) Initiating the death programme
Death receptors
Mitochondria

3) The Bcl-2 family
4) Stopping the death programme

Question 10

What are Caspases?

Answer 10

Cysteine-dependent aspartate-directed proteases

• Executioners of apoptosis
• Activated by proteolysis
• Cascade of activation

Question 11

What are the initiator and effector caspases?

Answer 11

CARD - CAspase Recruitment Domain
DED - Death Effector Domain

Initiator caspases: trigger apoptosis by cleaving and activating..

• caspase 2 (CARD)
• caspase 9 (CARD)
• caspase 10 (DED)
• caspase 8 (DED)

Effector caspases: carry out the apoptotic programme. Cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown). Activate enzymes (incl. protein kinases nucleases, e.g. Caspase-Activated DNase, CAD) by direct cleavage, or 
cleavage of inhibitory
molecules. 
 - caspase 3
 - caspase 6
 - caspase 7

Question 12

How is the active caspase formed?

Answer 12

Cleavage of the inactive procaspase precursor is followed by folding of 2 large and 2 small chains to form an active L2S2 heterotetramer

Question 13

Describe the caspase cascade

Answer 13

• amplification
• divergent responses
• regulation

see slide

Question 14

What are the mechanisms of caspase activation?

Answer 14

Death by design - Receptor-mediated (extrinsic) pathways

Death by default - Mitochondrial (intrinsic) death pathway.

Question 15

What are death receptors?

Answer 15

Secreted or transmembrane ligands (trimeric)

Question 16

Adaptor proteins death receptors?

Answer 16

?

Question 17

Describe the signalling through death receptors. e.g Fas/Fas-ligand

Answer 17

1. Receptor (Fas) trimerisation by ligand (Fas-L on lymphocyte)
2. Recruitment of adapter protein (FADD) through its DD to DD of Fas
3. Recruitment and oligomerisation of procaspase 8 through its DED to FADD DED –> Death-Inducing Signalling Complex (DISC)

Question 18

Describe what happens to procaspase 8?

Answer 18

• Some initiator procaspases have intrinsic low catalytic activity – oligomerisation allows transcleavage
• others are activated by conformational change on oligomerisation
• Need at least 2 procaspases to form an active tetramer

Initiator procaspase 8 oligomerisation results in cleavage and activation.

Question 19

What inhibits the activation of death receptors of caspase 8?

Answer 19

FLIP - caspase homology in DED domain, but no proteolytic activity therefore competes with procaspase

It Competes for binding to receptor tails / FADD via DED domains

Incorporates into receptor-procaspase complexes and interferes with transcleavage

Question 20

What is the function of caspase 8?

Answer 20

It activates downstream effector caspases